Posttranslational and Therapeutic Control of Gasdermin-Mediated Pyroptosis and Inflammation.

Pyroptosis is a proinflammatory form of cell death, mediated by membrane pore-forming proteins called gasdermins. Gasdermin pores allow the release of the pro-inflammatory cytokines IL-1ß and IL-18 and cause cell swelling and cell lysis leading to release of other intracellular proteins that act as alarmins to perpetuate inflammation. The best characterized, gasdermin D, forms pores via its N-terminal domain, generated after the cleavage of full length gasdermin D by caspase-1 or -11 (caspase-4/5 in humans) typically upon sensing of intracellular pathogens. Thus, gasdermins were originally thought to largely contribute to pathogen-induced inflammation. We now know that gasdermin family members can also be cleaved by other proteases, such as caspase-3, caspase-8 and granzymes, and that they contribute to sterile inflammation as well as inflammation in autoinflammatory diseases or during cancer immunotherapy. Here we briefly review how and when gasdermin pores are formed, and then focus on emerging endogenous mechanisms and therapeutic approaches that could be used to control pore formation, pyroptosis and downstream inflammation.

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts.

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Overexpression of iASPP is required for autophagy in response to oxidative stress in choriocarcinoma.

BACKGROUND: Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases developed from trophoblasts. ASPP (Ankyrin-repeat, SH3-domain and proline-rich region containing protein) family proteins, ASPP1 and ASPP2, have been reported to be dysregulated in GTD. They modulate p53 activities and are responsible for multiple cellular processes. Nevertheless, the functional role of the ASPP family inhibitory member, iASPP, is not well characterized in GTD. METHODS: To study the functional role of iASPP in GTD, trophoblastic tissues from normal placentas, hydatidiform mole (HM) and choriocarcinoma were used for immunohistochemistry, whereas siRNAs were used to manipulate iASPP expression in choriocarcinoma cell lines and study the subsequent molecular changes. RESULTS: We demonstrated that iASPP was overexpressed in both HM and choriocarcinoma when compared to normal placenta. Progressive increase in iASPP expression from HM to choriocarcinoma suggests that iASPP may be related to the development of trophoblastic malignancy. High iASPP expression in HM was also significantly associated with a high expression of autophagy-related protein LC3. Interestingly, iASPP silencing retarded the growth of choriocarcinoma through senescence instead of induction of apoptosis. LC3 expression decreased once iASPP was knocked down, suggesting a downregulation on autophagy. This may be due to iASPP downregulation rendered decrease in Atg5 expression and concomitantly hindered autophagy in choriocarcinoma cells. Autophagy inhibition per se had no effect on the growth of choriocarcinoma cells but increased the susceptibility of choriocarcinoma cells to oxidative stress, implying a protective role of iASPP against oxidative stress through autophagy in choriocarcinoma. CONCLUSIONS: iASPP regulates growth and the cellular responses towards oxidative stress in choriocarcinoma cells. Its overexpression is advantageous to the pathogenesis of GTD. (266 words).

The molecular, cellular and pathophysiological roles of iRhom pseudoproteases.

iRhom proteins are catalytically inactive relatives of rhomboid intramembrane proteases. There is a rapidly growing body of evidence that these pseudoenzymes have a central function in regulating inflammatory and growth factor signalling and consequent roles in many diseases. iRhom pseudoproteases have evolved new domains from their proteolytic ancestors, which are integral to their modular regulation and functions. Although we cannot yet conclude the full extent of their molecular and cellular mechanisms, there is a clearly emerging theme that they regulate the stability and trafficking of other membrane proteins. In the best understood case, iRhoms act as regulatory cofactors of the ADAM17 protease, controlling its function of shedding cytokines and growth factors. It seems likely that as the involvement of iRhoms in human diseases is increasingly recognized, they will become the focus of pharmaceutical interest, and here we discuss what is known about their molecular mechanisms and relevance in known pathologies.

Mechanistic insights into the evolution of DUF26-containing proteins in land plants.

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation. However, gene and genome duplications pose difficulties for functional characterization and translational research. Here we infer the evolutionary history of the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 emerged in secreted proteins. Domain duplications and rearrangements led to the appearance of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). The DUF26 is land plant-specific but structural analyses of PDLP ectodomains revealed strong similarity to fungal lectins and thus may constitute a group of plant carbohydrate-binding proteins. CRKs expanded through tandem duplications and preferential retention of duplicates following whole genome duplications, whereas PDLPs evolved according to the dosage balance hypothesis. We propose that new gene families mainly expand through small-scale duplications, while fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

Characteristics of candidate genes associated with embryonic development in the cow: Evidence for a role for WBP1 in development to the blastocyst stage.

The goal was to gain understanding of how 12 genes containing SNP previously related to embryo competence to become a blastocyst (BRINP3, C1QB, HSPA1L, IRF9, MON1B, PARM1, PCCB, PMM2, SLC18A2, TBC1D24, TTLL3 and WBP1) participate in embryonic development. Gene expression was evaluated in matured oocytes and embryos. BRINP3 and C1QB were not detected at any stage. For most other genes, transcript abundance declined as the embryo developed to the blastocyst stage. Exceptions were for PARM1 and WBP1, where steady-state mRNA increased at the 9-16 cell stage. The SNP in WBP1 caused large differences in the predicted three-dimensional structure of the protein while the SNP in PARM1 caused smaller changes. The mutation in WBP1 causes an amino acid substitution located close to a P-P-X-Y motif involved in protein-protein interactions. Moreover, the observation that the reference allele varies between mammalian species indicates that the locus has not been conserved during mammalian evolution. Knockdown of mRNA for WBP1 decreased the percent of putative zygotes becoming blastocysts and reduced the number of trophectoderm cells and immunoreactive CDX2 in the resulting blastocysts. WBP1 is an important gene for embryonic development in the cow. Further research to identify how the SNP in WBP1 affects processes leading to differentiation of the embryo into TE and ICM lineages is warranted.

[Genetic Control of Circadian Rhythms and Aging].

The review establishes a link between a group of genes which are conserved in evolution and form a molecular oscillator responsible for generation of circadian rhythms and genetic determinants of aging including associated pathways of intracellular signaling. An analysis of mechanisms of development of age-dependent pathologies is conducted from the viewpoint of circadian genetics. Systematic data of circadian gene expression studies in animals demonstrating different rates of aging from accelerated to negligible are presented.

Computational Analysis of the Binding Specificities of PH Domains.

Pleckstrin homology (PH) domains share low sequence identities but extremely conserved structures. They have been found in many proteins for cellular signal-dependent membrane targeting by binding inositol phosphates to perform different physiological functions. In order to understand the sequence-structure relationship and binding specificities of PH domains, quantum mechanical (QM) calculations and sequence-based combined with structure-based binding analysis were employed in our research. In the structural aspect, the binding specificities were shown to correlate with the hydropathy characteristics of PH domains and electrostatic properties of the bound inositol phosphates. By comparing these structure properties with sequence-based profiles of physicochemical properties, PH domains can be classified into four functional subgroups according to their binding specificities and affinities to inositol phosphates. The method not only provides a simple and practical paradigm to predict binding specificities for functional genomic research but also gives new insight into the understanding of the basis of diseases with respect to PH domain structures.

Protein marker profiling in different T classification in laryngeal squamous cell carcinoma.

BACKGROUND: The identification of specific biomarkers related to laryngeal squamous cell carcinoma (SCC) will be helpful in early detection and determination of reasonable treatment options, which are crucial for the prognosis of patients with laryngeal SCC. The purpose of this study was to profile the protein markers that can be used for diagnosis and prognosis of laryngeal SCC and to observe laryngeal SCC progression with distinct stages of malignant transformation. METHODS: Two hundred twenty-five proteins were tested in 84 pairs of tumors and adjacent nontumor mucosa samples using protein pathway arrays (PPAs). Ingenuity pathway analysis (IPA) enrichment analysis was performed and protein expression profiles in different T classification were mapped by grid analysis of time-series expression (GATE). RESULTS: Among 16 proteins differently expressed between tumors and normal tissues, we selected 9 proteins (TTF-1, CDK2, Eg5, proliferating cell nuclear antigen [PCNA], Bcl-xL, 14-3-3ß, p27, SRC-1, cytokeratin 18) as markers for classification. From the IPA analysis, we observed a more malignant transformation from T3 to T4 at the protein level and described the changing patterns of the proteins' expression in this progression. JAK2, keratin 10, and IL-3Rα were identified as markers for prognosis. The risk model based on histological grade, T classification, N classification, JAK2, and IL-3Rα can predict the prognosis with 85.5% accuracy. CONCLUSION: This study indicated that dysregulated signaling proteins can be selected as useful biomarkers for tumor classification and predicting the outcome in patients with laryngeal SCC. The changing patterns of the proteins' expression in different stages were related to the more malignant transformation and further studies will focus on the role of these proteins in laryngeal SCC progression.

Comparative purification and characterization of two HIN domains, hematopoietic interferon-inducible nuclear antigens with a 200-amino-acid repeat, in murine AIM2-like receptors.

HIN (hematopoietic interferon-inducible nuclear antigens with a 200-amino-acid repeat) domains are found in all AIM2-like receptors (ALRs) and mediate protein/DNA interactions to regulate innate immunity. We cloned, expressed, and purified the human interferon-inducible protein p202 (Ifi202) HINb domain and the murine Ifi203 HIN domain using Escherichia coli JM109 (DE3) host cells. The Ifi203 HIN domain is a monomer in solution, and it has the ability to bind both double-stranded DNA and RNA. In contrast, the Ifi202 HINb domain is a dimer in solution, and no interaction with double-stranded DNA or RNA was detected. A complex of the Ifi203 HIN domain and double-stranded DNA was prepared, and crystals of the complex were obtained. To analyze further the dimeric interface of the Ifi202 HINb domain and the DNA binding site of the Ifi203 HIN domain, models of both proteins were developed. This work provides a basis for understanding the structure and function of HIN domains.

JAV1 controls jasmonate-regulated plant defense.

Plants evolve effective mechanisms to protect themselves from environmental stresses and employ jasmonates as vital defense signals to defend against insect attack and pathogen infection. Jasmonates are also recognized as an essential growth regulator by which diverse developmental processes are mediated. Despite substantial research, there are no key signaling components reported yet to control jasmonate-regulated plant defense independent of developmental responses. We identify JAV1, a key gene in the jasmonate pathway, which functions as a negative regulator to control plant defense but does not play a detectable role in plant development. Our results suggest that when encountering insect attack and pathogen infection, plants accumulate jasmonates that trigger JAV1 degradation via the 26S proteasome to activate defensive gene expression and elevate resistances against both insects and pathogens. These findings have provided insight into the molecular mechanism by which plants integrate jasmonate signals to protect themselves from insect attack and pathogen infection.

Dissection of the dimerization modes in the DJ-1 superfamily.

The DJ-1 superfamily (DJ-1/ThiJ/PfpI superfamily) is distributed across all three kingdoms of life. These proteins are involved in a highly diverse range of cellular functions, including chaperone and protease activity. DJ-1 proteins usually form dimers or hexamers in vivo and show at least four different binding orientations via distinct interface patches. Abnormal oligomerization of human DJ-1 is related to neurodegenerative disorders including Parkinson's disease, suggesting important functional roles of quaternary structures. However, the quaternary structures of the DJ-1 superfamily have not been extensively studied. Here, we focus on the diverse oligomerization modes among the DJ-1 superfamily proteins and investigate the functional roles of quaternary structures both computationally and experimentally. The oligomerization modes are classified into 4 types (DJ-1, YhbO, Hsp, and YDR types) depending on the distinct interface patches (I-IV) upon dimerization. A unique, rotated interface via patch I is reported, which may potentially be related to higher order oligomerization. In general, the groups based on sequence similarity are consistent with the quaternary structural classes, but their biochemical functions cannot be directly inferred using sequence information alone. The observed phyletic pattern suggests the dynamic nature of quaternary structures in the course of evolution. The amino acid residues at the interfaces tend to show lower mutation rates than those of non-interfacial surfaces.

Somitogenesis in the anole lizard and alligator reveals evolutionary convergence and divergence in the amniote segmentation clock.

The axial skeleton is a defining feature of vertebrates and is patterned during somitogenesis. Cyclically expressed members of the notch and other signaling pathways, described as the 'segmentation clock', regulate the formation of somite boundaries. Comparisons among vertebrate model systems have revealed fundamental shifts in the regulation of expression among critical genes in the notch pathway. However, insights into the evolution of these expression differences have been limited by the lack of information from non-avian reptiles. We analyzed the segmentation clock of the first Lepidosaurian reptile sequenced, the green anole lizard, Anolis carolinensis, for comparison with avian and mammalian models. Using genomic sequence, RNA-Seq transcriptomic data, and in situ hybridization analysis of somite-stage embryos, we carried out comparative analyses of key genes and found that the anole segmentation clock displays features common to both amniote and anamniote vertebrates. Shared features with anamniotes, represented by Xenopus laevis and Danio rerio, include an absence of lunatic fringe (lfng) expression within the presomitic mesoderm (PSM), a hes6a gradient in the PSM not observed in the chicken or mouse, and EGF repeat structure of the divergent notch ligand, dll3. The anole and mouse share cycling expression of dll1 ligand in the PSM. To gain insight from an Archosaurian reptile, we analysed LFNG and DLL1 expressions in the American alligator. LFNG expression was absent in the alligator PSM, like the anole but unlike the chicken. In contrast, DLL1 expression does not cycle in the PSM of the alligator, similar to the chicken but unlike the anole. Thus, our analysis yields novel insights into features of the segmentation clock that are evolutionarily basal to amniotes versus those that are specific to mammals, Lepidosaurian reptiles, or Archosaurian reptiles.

The evolutionary history of YAP and the hippo/YAP pathway.

The Hippo/YAP pathway plays an important role in animal organ size control, which it exerts by regulating tissue proliferation and apoptosis rates as a response to developmental cues, cell contact, and density. With the ever increasing advance in genome sequencing and analysis tools, our understanding of the animal world and its evolution has greatly increased in the recent years. We used bioinformatic tools to study the evolution of the Hippo/YAP pathway focusing on the transcriptional coactivator YAP, which is a pivotal effector of the pathway. The aim was to establish the origin and mode of development of YAP and its pathway in the animal world. Some pathway members can be already identified in single-celled eukaryotes like the yeast that have preceded multicellular animals. Interestingly, we can find most of the components that are present in human in the sea-anemone Nematostella, which belongs to a very basal group of metazoans, the cnidarians. All the major domains of YAP have been conserved between cnidarians and mammals, and YAP can be identified even in the more basal placozoan clade. We show a very high degree of conservation in regions such as the WW and the TEAD-binding domains, TEAD being the major DNA-binding partner of YAP. Remarkably, we found that the location of an intron in the WW1 genomic region has been invariant along an evolutionary span of over 700 My. We have followed the evolutionary changes in YAP and in other main components of the pathway from the first metazoans such as sponges, described the phylogenetic relationships between the YAP genes and indicated where YAP and other components have been secondarily lost. Evidence is provided that YAP and its binding partner TEAD demonstrate strong coevolution. This gives further support for the importance of the TEAD-YAP association. Beyond contributing to an understanding of the evolutionary history of this pathway, we have provided insights into the "birth" of this pathway, its functions and its mode of operation in animals with different body plans, development, and life styles.

Docking proteins.

Docking proteins comprise a distinct category of intracellular, noncatalytic signalling protein, that function downstream of a variety of receptor and receptor-associated tyrosine kinases and regulate diverse physiological and pathological processes. The growth factor receptor bound 2-associated binder/Daughter of Sevenless, insulin receptor substrate, fibroblast growth factor receptor substrate 2 and downstream of tyrosine kinases protein families fall into this category. This minireview focuses on the structure, function and regulation of these proteins.

Characterization and transcriptional regulation analysis of the porcine TNFAIP8L2 gene.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a new member of the tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) family that functions as an important factor in the maintenance of immune homeostasis. In this study, we cloned the cDNA sequences and analyzed the genomic structure of porcine TNFAIP8L2. RH mapping using the IMpRH panel showed that this gene was closely linked to microsatellite marker SW512 in pig chromosome 4. Subcellular localization analysis showed GFP-TNFAIP8L2 fusion protein distributed in nucleus as well as cytoplasm including mitochondria and endoplasmic reticulum. Real-time PCR analysis revealed that porcine TNFAIP8L2 was more highly expressed in spleen than other tissues. To understand its characterization of transcriptional regulation, we cloned approximately 2 kb of 5'-regulatory region upstream to the porcine TNFAIP8L2 translational start site and generated sequential deletion constructs evaluated in dual-luciferase reporter assay. The results demonstrated that its core promoter is 435 base pairs (bp) upstream to the transcription initiation site. Then, site-directed mutation experiment combined with electrophoretic mobility shift assay (EMSA) indicated that M-CAT binding factor (MCBF) and activator protein 1 (AP-1) were important transcription factors for porcine TNFAIP8L2. These findings provide an important basis for further understanding of porcine TNFAIP8L2 regulation and function in swine.

Diversity of lysophosphatidic acid receptor-mediated intracellular calcium signaling in early cortical neurogenesis.

Lysophosphatidic acid (LPA) is a membrane-derived lysophospholipid that can induce pleomorphic effects in neural progenitor cells (NPCs) from the cerebral cortex, including alterations in ionic conductance. LPA-induced, calcium-mediated conductance changes have been reported; however, the underlying molecular mechanisms have not been determined. We show here that activation of specific cognate receptors accounts for nearly all intracellular calcium responses evoked by LPA in acutely cultured nestin-positive NPCs from the developing mouse cerebral cortex. Fast-onset changes in intracellular calcium levels required release from thapsigargin-sensitive stores by a pertussis toxin-insensitive mechanism. The influx of extracellular calcium through Cd(2+)/Ni(2+)-insensitive influx pathways, approximately one-half of which were Gd(3+) sensitive, contributed to the temporal diversity of responses. Quantitative reverse transcription-PCR revealed the presence of all five known LPA receptors in primary NPCs, with prominent expression of LPA(1), LPA(2), and LPA(4). Combined genetic and pharmacological studies indicated that NPC responses were mediated by LPA(1) (approximately 30% of the cells), LPA(2) (approximately 30%), a combination of receptors on single cells (approximately 30%), and non-LPA(1,2,3) pathways (approximately 10%). LPA responsivity was significantly reduced in more differentiated TuJ1(+) cells within cultures. Calcium transients in a large proportion of LPA-responsive NPCs were also initiated by the closely related signaling lipid S1P (sphingosine-1-phosphate). These data demonstrate for the first time the involvement of LPA receptors in mediating surprisingly diverse NPC calcium responses involving multiple receptor subtypes that function within a single cell. Compared with other known factors, lysophospholipids represent the major activator of calcium signaling identified within NPCs at this early stage in corticogenesis.

Orexins and gastrointestinal functions.

Orexin A (OXA) and orexin B (OXB) are recently discovered neuropeptides that appear to play a role in various distinct functions such as arousal and the sleep-wake cycle as well as on appetite and regulation of feeding and energy homeostasis. Orexins were first described as neuropeptides expressed by a specific population of neurons in the lateral hypothalamic area, a region classically implicated in feeding behaviour. Orexin neurons project to numerous brain regions, where orexin receptors have been shown to be widely distributed: both OXA and OXB act through two subtypes of receptors (OX1R and OX2R) that belong to the G protein-coupled superfamily of receptors. Growing evidence indicates that orexins act in the central nervous system also to regulate gastrointestinal functions: animal studies have indeed demonstrated that centrally-injected orexins or endogenously released orexins in the brain stimulates gastric secretion and influence gastrointestinal motility. The subsequent identification of orexins and their receptors in the enteric nervous system (including the myenteric and the submucosal plexuses) as well as in mucosa and smooth muscles has suggested that these neuropeptides may also play a local action. In this view, emerging studies indicate that orexins also exert region-specific contractile or relaxant effects on isolated gut preparations. The aim of the proposed review is to summarize both centrally- and peripherally-mediated actions of orexins on gastrointestinal functions and to discuss the related physiological role on the basis of the most recent findings.

Hypocretin/orexin contributes to the expression of some but not all forms of stress and arousal.

Hypocretin/orexin has a well-established role in wakefulness and in the maintenance of arousal. Because stress is associated with arousal, it has been proposed that hypocretin is also involved in stress. However, it is not clear if this is true for all forms of stress. To clarify this issue, we compared four conditions combining high arousal with no or low stress (wakefulness and exploration) or high stress (contextual fear and restraint) in the rat. We looked at Fos expression in hypocretin neurons, hypocretin-1 levels in cerebrospinal fluid and cardiovascular and behavioural changes after pharmacological blockade with the dual hypocretin receptor antagonist, almorexant. Fos expression in hypocretin neurons was highest with wakefulness and exploration, also high with fear but not significant with restraint. Hypocretin-1 levels were consistent with this pattern, although the differences were not as marked. Hypocretin receptor blockade with almorexant reduced the pressor, tachycardic and locomotor responses of wakefulness and exploration as well as the pressor and sympathetic component of the tachycardic response of fear. In contrast, almorexant did not reduce the pressor and tachycardic responses of restraint and nor did it reduce the pressor, tachycardic and locomotor responses of another stressor, i.e. cold exposure. Thus, hypocretin is not involved in all forms of stress. Comparison of the different conditions suggests that, regardless of stress, hypocretin involvement occurs when the arousal associated with the response includes increased attention to environmental cues. When it does, hypocretin will at least contribute to the cardiovascular response. The findings are of clinical relevance to some forms of psychological stress.

TGF-beta signaling proteins and the Protein Ontology.

BACKGROUND: The Protein Ontology (PRO) is designed as a formal and principled Open Biomedical Ontologies (OBO) Foundry ontology for proteins. The components of PRO extend from a classification of proteins on the basis of evolutionary relationships at the homeomorphic level to the representation of the multiple protein forms of a gene, including those resulting from alternative splicing, cleavage and/or post-translational modifications. Focusing specifically on the TGF-beta signaling proteins, we describe the building, curation, usage and dissemination of PRO. RESULTS: PRO is manually curated on the basis of PrePRO, an automatically generated file with content derived from standard protein data sources. Manual curation ensures that the treatment of the protein classes and the internal and external relationships conform to the PRO framework. The current release of PRO is based upon experimental data from mouse and human proteins wherein equivalent protein forms are represented by single terms. In addition to the PRO ontology, the annotation of PRO terms is released as a separate PRO association file, which contains, for each given PRO term, an annotation from the experimentally characterized sub-types as well as the corresponding database identifiers and sequence coordinates. The annotations are added in the form of relationship to other ontologies. Whenever possible, equivalent forms in other species are listed to facilitate cross-species comparison. Splice and allelic variants, gene fusion products and modified protein forms are all represented as entities in the ontology. Therefore, PRO provides for the representation of protein entities and a resource for describing the associated data. This makes PRO useful both for proteomics studies where isoforms and modified forms must be differentiated, and for studies of biological pathways, where representations need to take account of the different ways in which the cascade of events may depend on specific protein modifications. CONCLUSION: PRO provides a framework for the formal representation of protein classes and protein forms in the OBO Foundry. It is designed to enable data retrieval and integration and machine reasoning at the molecular level of proteins, thereby facilitating cross-species comparisons, pathway analysis, disease modeling and the generation of new hypotheses.